Phanerochaete chrysospoirium strains CNCM I-1511, I-1512 and I-1513 for producing lignin peroxidase and manganese peroxidase

ABSTRACT

PCT No. PCT/FR95/01746 Sec. 371 Date Jul. 1, 1997 Sec. 102(e) Date Jul. 1, 1997 PCT Filed Dec. 28, 1995 PCT Pub. No. WO96/21008 PCT Pub. Date Jul. 11, 1996Phanerochaete chrysosporium strains CNCM numbers I-1511, I-1512 and I-1513 are found that produce increased amounts of manganese peroxidase (MnP) and lignin peroxidase (LiP). Immobilized cells may be used for culturing, and culture media is preferably supplemented with a phospholipid as a carbon source and/or veratryl alcohol to stabilize enzymes produced. Culturing conditions can be selected to modify the MnP/LiP ratio in favor of the production of MnP or LiP.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a process for producing peroxidasesfrom the fungus Phanerochaete chrysosporium.

2. Description of the Background

Phanerochaete chrysosporium is a fungus associated with "white rot ofwood". It is a hymenomycete which belongs to the order Aphylophoralesand to the family Corticaceae. It has the property of degrading ligninto the point of mineralization (final products: CO₂ +H₂ O).

This fungus produces exocellular peroxidases: the latter comprise, inparticular, isozymes of manganese peroxidase (MnPs) [KUWAHARA et al.FEBS Let., 169, pp. 247-250, (1984)] and those of lignin peroxidase(LiPs) [TIEN M. and KIRK T. K., Science, 221, pp. 661-663, (1983); GLENNet al. Biochem. Biophys. Res. Commun., 114, pp. 1077-1083]. Theseenzymes are glycosylated haemoproteins whose average molecular mass is40 kDa [LEISOLA et al., J. Biol. Chem., 262 pp. 419-424, (1984)].

Manganese peroxidases and lignin peroxidases are capable of catalysingthe oxidation of numerous aromatic substrates, especially lignin, usinghydrogen peroxide as cosubstrate. These properties find their mainapplications in the field of papermaking and that of waste treatment.

Hitherto, the lignin peroxidases are the ones which have chiefly beenused in this type of application.

For example, French Patent 2,574,427 describes two strains ofPhanerochaete chrysosporium possessing especially high lignin peroxidaseactivity, and their culture on a medium containing an assimilablenitrogen source as well as an assimilable carbon source and a source ofassimilable inorganic salts.

The proposal has also been made (French Patent 2,600,077) to culturePhanerochaete chrysosporium on a basal culture medium supplemented withunsaturated fatty acids and/or natural amino acids. Other teams haveproposed adding detergent of the Tween type [FAISON and KIRK, Appl.Environ. Microbiol. (1985), 49, pp. 299-304], or alternatively addingveratryl alcohol [LEISOLA et al., J. Biotechnol. (1985), 3, pp. 97-107],in order to increase the synthesis of lignin peroxidase.

The inventors' team has also, in previous investigations, demonstratedvarious parameters whose optimization enabled the production of ligninperoxidase to be increased: addition of oleic acid and/or of exogenousphospholipids, control of the culture temperature, and the like. Theseinvestigations led to the development of a process for producing ligninperoxidase, which forms the subject of European Patent 0,437,500.

This process comprises several successive steps, each of them beingperformed under different culture conditions; the first step isperformed on synthetic medium comprising inorganic salts, a carbonsource and a nitrogen source, in the presence of yeast extract, a sourceof phospholipids and emulsified fatty acids; the mycelium formed is thencultured in a partially renewed culture medium, supplemented withveratryl alcohol but not comprising emulsified fatty acids, and whosephospholipid content represents only 1/7 to 1/8 of that of the culturemedium used in the first step; in a third step, the medium is replacedcompletely by a new medium, comprising the same proportion ofphospholipids and of veratryl alcohol as that of the second step, andthe carbon source, the nitrogen source and the yeast extract at 1/4 oftheir content in the medium of the first step; lastly, a fourth culturestep is performed in a medium lacking yeast extract, carbon source andemulsified fatty acids, but comprising the same proportion ofphospholipids and of veratryl alcohol as that of the preceding steps.

The lignin peroxidase may then be recovered from the culture medium. Theuse of this process enables the enzyme content to be increased verysignificantly; the lignin peroxidase activity in the medium isapproximately 240 U per liter and per day.

The research performed hitherto has mainly been concerned withincreasing the lignin peroxidase activity. However, the manganeseperoxidases are being seen increasingly to be capable of performing akey role in the bioconversion of polymeric aromatic compounds of thelignin type. In effect, contrary to lignin peroxidases whose activity islimited by the low penetration of the lignified walls by the enzyme,manganese peroxidases act via species of low molecular weight whichdiffuse readily. Briefly, the catalytic cycle of manganese peroxidasesinvolves the oxidation of Mn(II) to Mn(III) which, after complexing withorganic acids, generates diffusing oxidizing species capable ofdepolymerizing natural lignin [WARIISHI K., et al., Biochem. Biophys.Res. Comm. 176, pp. 269-276, (1991)].

BONNARME and JEFFRIES [J. Ferment. Bioeng. 70:158-163 (1990)] studiedthe regulation of the production of Phanerochaete chrysosporium ligninperoxidase and manganese peroxidase under different culture conditions;they observed in this way that the amount of these enzymes variedespecially in accordance with the Mn(II) concentration: at lowconcentration, lignin peroxidase is produced preferentially (760nmol/ml.min under optimal conditions), while at high concentration, theproduction of manganese peroxidase (950 nmol/ml.min under optimalconditions) is the one which is favoured.

SUMMARY OF THE INVENTION

It was the objective of the inventors to increase the production ofPhanerochaete chrysosporium exocellular peroxidases from cultures ofthis fungus, and also to increase the MnP/LiP ratio.

For this purpose, the inventors succeeded in obtaining new strains(hereinafter designated MIC 390, MIC 249 and MIC 396) which arehypersecretory of the exocellular peroxidases lignin peroxidase andmanganese peroxidase. In addition, they devised specific cultureconditions which can be carried out either with free cells or with cellsimmobilized on a support. This enables the yield of lignin peroxidaseand manganese peroxidase to be increased very significantly relative tothat obtained with the cultures of the prior art: for example, thecultures obtained according to the invention can produce approximatelytwice as much lignin peroxidase as those of Patent EP 0,437,500, and 10times as much manganese peroxidase as those obtained previously byBONNARME and JEFFRIES.

Implementation of the present invention makes it possible, in addition,to control and modify the manganese peroxidase/lignin peroxidase ratioaccording to requirements.

DETAILED DESCRIPTION OF THE INVENTION

The subject of the present invention is Phanerochaete chrysosporiumstrains MIC 249, MIC 390 and MIC 396, which were deposited on 20thDecember 1994 with the CNCM (Collection Nationale de Cultures deMicroorganismes [National Collection of Microorganism Cultures]) held bythe Pasteur Institute, 26 rue du Docteur Roux in Paris, under therespective numbers I-1511, I-1512 and I-1513.

The subject of the present invention is also a process for producinglignin peroxidase and/or manganese peroxidase from a culture ofPhanerochaete chrysosporium, which process is characterized in that itcomprises the culturing of at least one Phanerochaete chrysosporiumstrain chosen from the group consisting of the strains MIC 249, MIC 390and MIC 396 mentioned above.

These cultures are produced, according to the standard techniques ofculture of Phanerochaete chrysosporium which are known per se, from aninoculum consisting of mycelial fragments obtained from a preculture orfrom spores (2×10⁵ spores per ml approximately).

The culture medium comprises at least one carbon source, at least onenitrogen source, inorganic salts, trace elements and vitamins, and it issupplemented with yeast extract.

The concentration of the carbon source is preferably between 5 and 20g/l.

A carbon source preferentially used comprises glycerol, but other carbonsources which are assimilable more slowly or more rapidly can be used,alone or mixed; examples of such carbon sources include maltose,raffinose, starch, xylose, rhamnose, arabinose, fructose, sorbitol,mannose, cellobiose and cellulose.

According to a preferred embodiment of the process according to theinvention, the carbon source comprises at least one phospholipid chosenfrom the group consisting of phosphatidylcholine (PC),lysophosphatidylcholine (LPC), phosphatidylethanolamine (PE),acylphosphatidylethanolamine (APE), phosphatidylinositol (PI) andphosphatidic acid (PA), or a mixture of the said phospholipids; themixture can, in this case, be a reconstituted mixture of phospholipids,or alternatively a mixture of phospholipids originating from a naturalsource, for example soya phospholipids. Advantageously, if a mixture ofphospholipids is used, it comprises more than 25% ofphosphatidylinositol and less than 15% of PC.

Preferably, the concentration of the phospholipid or mixture ofphospholipids (reconstituted, or originating from soya) is between 0.1and 10 g/liter.

The nitrogen source can, for example, consist of amino acids, sodiumnitrate or a mixture of these different nitrogen sources, in combinationwith yeast extract. The concentration of the nitrogen source ispreferably between 0.5 and 20 g/l.

The assimilable inorganic salts comprise potassium, calcium andmagnesium salts, and are used at a concentration of between 0.5 and 100mM.

The trace elements are mainly composed of iron sulphate, zinc sulphate,manganese sulphate and copper sulphate. The concentrations of iron, zincand copper sulphates are the concentrations customarily used forculturing Phanerochaete chrysosporium (see, for example, European Patent0,437,500). The Mn²⁺ concentration has no influence on the production ofmanganese peroxidase under the culture conditions according to theinvention, and can vary over a relatively wide range; it is preferablybetween 1 and 600 mM.

The medium is also supplemented with a source of vitamins; it ispossible, for example, to use a mixture of vitamins whose compositionconforms to that given by TATUM et al. [Am. J. Bot., 37:38-46 (1950)].The mixture of vitamins is used at a concentration of between 0.001 gand 1 g per liter.

Moreover, the addition of veratryl alcohol (approximately 0.4 mM)enables the stability of the enzymes produced to be maintained.

According to a preferred embodiment of the present invention, theculture medium is, in addition, supplemented during culture by adding,in the proportion of 0.001 to 10 grammes per liter, at least onecomponent which activates the production of enzymes and/or protects thelatter; this activating and/or protective component can consist of oneof the constituents of the initial medium, or of a mixture of several ofthem.

Preferably, this activating and/or protective component comprisesveratryl alcohol, which is, in this case, used at a concentration ofbetween 0.1 and 1 gramme per liter. It is also possible to add asupplement of a phospholipid source rich in phosphatidylinositol, whichis, in this case, used in the proportion of 0.1 to 3 grammes per liter.

This addition may be performed, in the form of a solution, emulsion orliposomes, by adding a mixture of surfactant (Tween 80 for example) withfatty acids (C18:12, C18:2) and phospholipids rich inphosphatidylinositol.

The supplementation of the medium may be performed continuously (pump)or discontinuously.

The supplementation with activating and/or protective component(s) isperformed when the culture has reached a growth stage such that theexpression of the genes coding for the peroxidases (production of themessenger RNAs coding for the manganese peroxidases and ligninperoxidases) is beginning to take place. This stage, which has beendefined experimentally, is reached after a period which can varyaccording to the strains and the culture conditions, but which generallyrepresents approximately 2 days of culture at a temperature of between28° C. and 40° C.

To carry out the process according to the invention, culturing may beperformed in a manner known per se, either with cells immobilized on asupport or with free cells.

In the case of immobilized cells, the latter are coupled to or adsorbedon one or several hydrophobic, hydrophilic or neutral supports, thesurface of which is preferably rough, and containing, for example,cavities, meshing (gauze or web), hollows or holes. These supports,hollow or otherwise, are arranged in ordered fashion or otherwise, fixedor mobile in the liquid phase consisting of the appropriate nutrientmedium, and can have a variety of shapes (cylindrical or cubic, in oneor several pieces). These supports, like the medium, may be replacedand/or renewed continuously or discontinuously during culture.

As non-limiting examples of unordered fixed supports, there may bementioned cylindrical Raschig rings made of stoneware, glass, metal orplastic; polyurethane, polyester and polyamide foams; metal or plasticfilings and plastic thread.

As non-limiting examples of ordered fixed supports, there may bementioned gauze or meshing: stainless steel, glass, plastic,polyurethane, polyester, nylon, polyacrylate, polyamide, and the like.

As non-limiting examples of supports which are mobile in the liquidphase, there may be mentioned polyurethane, polyester, polyamide andextruded plastic supports.

In the case of free cells, the mycelium is in the form of balls whichcan be 0.5 to 5 mm in diameter. The bioreactors employed are of thebubble column or airlift type (diameter/height ratio d/h=1/4 to 1/6approx.) or conventional type (d/h=1 to 1/2 approx.), and possess aspecific or non-specific stirrer module, for example: Rushton,marine-type propeller, MIG, or single or double helical band.

Advantageously, culturing is performed with aeration and agitation ofthe medium.

Aeration of the medium is carried out by introducing air, pure oxygen orany other mixture of gases providing a sufficient supply of oxygen tothe microorganism, by means of a device permitting homogeneousdispersion of this gas (sintered glass, rod).

Agitation of the medium may be performed mechanically. It can also beobtained pneumatically by direct action of the aeration system, or by anequivalent system used simultaneously.

The level of agitation and/or of aeration is chosen so as to permit ahomogeneous initial occupation of the support in the case of immobilizedcells, and the formation of mycelial balls of average diameter 0.5 to 5mm in the case of free cells, while limiting the shearing stressesundergone by the mycelial hyphae. This level may be variable during theculture period.

Advantageously, in the initial period (approximately 2 days), incubationtakes place at a temperature of approximately 37° C, and can be followedby a temperature change favourable to the production of peroxidases, asdescribed, for example, in Patent EP 0,437,500.

The production of manganese peroxidase and/or that of lignin peroxidase,and consequently the MnP/LiP ratio, can be controlled in accordance withthe age of the culture and/or the presence of activators, as well as thestrains used. It is thus possible to obtain enzyme cocktails whichpredominate either in manganese peroxidase or in lignin peroxidase(activity greater than or equal to 60% of the collective manganeseperoxidase and lignin peroxidase activities).

According to a variant of the process according to the invention, it ispossible, in addition, to control the MnP/LiP ratio, and to increasepreferentially either the production of manganese peroxidase or theproduction of lignin peroxidase. This variant is characterized in that:

to modify the MnP/LiP ratio in favour of the production of manganeseperoxidase, culturing of Phanerochaete chrysosporium is carried out fora period of more than 24 hours and less than 90 hours in the presence ofa mixture of phospholipids rich in phosphatidylinositol, as is definedabove, at a concentration of between 0.5 and 5 g/l, preferably of theorder of 1.5 g/l;

to modify the MnP/LiP ratio in favour of the production of ligninperoxidase, culturing of Phanerochaete chrysosporium is carried out fora period of more than 90 hours and less than 350 hours in the presenceof a mixture of phospholipids, as is defined above, at a concentrationof between 0.5 and 5 g/l.

Depending on the applications, the culture liquors thus constitutingenzyme cocktails more or less enriched in manganese peroxidase or inlignin peroxidase may be used directly, or after concentration, forexample by ultrafiltration. Where appropriate, purification on a MONO-Qtype column (Pharmacia Biotech S.A.; France) is carried out.

A better understanding of the present invention will be gained from thefurther description which follows, which refers to examples ofembodiment of the process according to the invention.

It should be clearly understood, however, that these examples are givenonly by way of illustration of the subject of the invention, of whichthey in no way constitute a limitation.

I--Use of Hypersecretory Strains

The strains which were selected according to the invention are thePhanerochaete chrysosporium strains MIC 249 (CNCM I-1511), MIC 390 (CNCMI-1512) and MIC 396 (CNCM I-1513).

EXAMPLE 1

Comparison of the Production of Lignin Peroxidases and of ManganesePeroxidases by Different Strains

The production capacity of the hypersecretory strains according to theinvention, MIC 249, MIC 390 and MIC 396, relative to the referencestrain BKM-F-1767 (ATCC 24725), is verified on cultures set up in 250-mlErlenmeyers under the following conditions:

Composition of the medium

Glycerol 10 g/l

Disodium tartrate 2.3 g/l

Diammonium tartrate 1.842 g/l

KH₂ PO₄ 2 g/l

CaCl₂.2H₂ O 0.14 g/l

MgSO₄.7H₂ O 0.70 g/l

FeSO₄.7H₂ O 0.07 g/l

ZnSO₄.7H₂ O 0.046 g/l

MnSO₄.H₂ O 0.035 g/l

CUSO₄.5H₂ O 0.007 g/l

Yeast extract 1 g/l

Nat89 0.5 g/l

Veratryl alcohol 0.42 g/l

Culture conditions

The medium is distributed under sterile conditions in the proportion of100 ml per Erlenmeyer; each Erlenmeyer contains cubes of polyurethanefoam.

Inoculation is carried out using mycelial fragments from a preculture.

Oxygenation of the Erlenmeyers is carried out at the start of culturing(T0), and incubation at 37° C is then carried out with stirring at 90 or120 rpm.

From 48 h of culture, daily oxygenation of the Erlenmeyers is carriedout, and incubation is continued at 37° C with stirring at 90 or 120rpm.

The lignin peroxidase and manganese peroxidase activities are measuredafter 4 days of culture according to the following protocols:

a) determination of lignin peroxidase activity: this activity isdetermined by measuring the rate of oxidation of veratryl alcohol to thecorresponding aldehyde in the presence of hydrogen peroxide [TIEN andKIRK, Proc. Natl. Acad. Sci. U.S.A. 81:2280-2284 (1984)]. The reactionis monitored at 30° C. by spectrophotometry at 310 nm. The molarextinction coefficient of veratraldehyde at this wavelength is 9300M⁻¹.cm⁻¹.

The activity in the medium is expressed in nkatal.ml⁻¹ or alternativelyin units/liter (U/l): one lignin peroxidase unit corresponds to onemicromole of veratraldehyde formed per minute.

b) determination of manganese peroxidase activity: this activity isdetermined by measuring the rate of oxidation of vanillylacetone in thepresence of MnSO₄ [PASZCZYNSKI et al. FEMS Microbiol. Lett., 29:37-41(1985)]. The reaction is monitored at 30° C. by spectrophotometry at 334nm. The molar extinction coefficient of vanillylacetone at thiswavelength is 18300 M⁻¹.cm⁻¹.

The activity in the medium is expressed in nkatal.ml⁻¹ or alternativelyin units/liter (U/l): one manganese peroxidase unit corresponds to onemicromole of vanillylacetone formed per minute.

The results are summarized in Table I below:

                  TABLE I                                                         ______________________________________                                                     MnP ACTIVITY                                                                              LiP ACTIVITY                                         STRAIN             (U/l)             (U/l)                                    ______________________________________                                        ATCC 24725   1200         900                                                 MIC 249           4800                 4200                                   MIC 390           5520                 2040                                   MIC 396           3500                 3150                                   ______________________________________                                    

These results show that, under these conditions, the strain I-1511 (MIC249) produces 4 times as much manganese peroxidase and lignin peroxidaseas the reference strain BKM-F-1767; the strain I-1512 (MIC 390) producesapproximately 5 times as much manganese peroxidase and twice as muchlignin peroxidase as the strain BKM-F-1767; and the strain I-1513 (MIC396) produces twice as much manganese peroxidase and lignin peroxidaseas the strain BKM-F-1767.

II--Influence of the Composition of the Medium and the CultureConditions on the Production of Lignin Peroxidases and of ManganesePeroxidases

EXAMPLE 2

Influence of the Age of the Culture

The strain MIC 249 is cultured under conditions identical to thosedescribed above in Example 1. Incubation is performed at 37° C. withstirring at 90 rpm.

The lignin peroxidase and manganese peroxidase activities are measuredat various culture times, and the MnP/LiP ratio is calculated. Theresults are summarized in Table II below.

                  TABLE II                                                        ______________________________________                                        AGE OF THE                                                                             MnP ACTIVITY  LiP ACTIVITY                                                                              MnP/LiP                                    CULTURE (h)                                                                             (U/l)                (U/l)                                                                                     RATIO                              ______________________________________                                        63       3270          1746        1.87                                       109.5            2694           2634                                                                                      1.02                              229                2346                                                                                       4290                                                                                      0.55                              ______________________________________                                    

EXAMPLE 3

Influence of the Phospholipid Concentration

The strain MIC 390 is cultured under the following conditions, on abasal medium whose composition is as follows:

Glycerol 10 g/l

Disodium tartrate 2.3 g/l

Diammonium tartrate 1.842 g/l

KH₂ PO₄ 2 g/l

CaCl₂.2H₂ O 0.14 g/l

MgSO₄.7H₂ O 0.70 g/l

FeSO₄.7H₂ O 0.07 g/l

ZnSO₄.7H₂ O 0.046 g/l

MnSO₄.H₂ O 0.035 g/l

CUSO₄.5H₂ O 0.007 g/l

Yeast extract 1 g/l

This basal medium is supplemented with a mixture of phospholipids,NAT89, at concentrations of between 0.5 g/l and 1.89 g/l.

NAT 89 is supplied by the company NATTERMAN PHOSPHOLIPID GmbH (Cologne,Germany), and its composition is as follows:

12% of phosphatidylcholine and lysophosphatidylcholine;

31% of phosphatidylethanolamine and acylphosphatidylethanolamine;

27% of phosphatidylinositol;

30% of phophatidic acid.

Culturing is performed as described in Example 1. Incubation isperformed at 37° C. with stirring at 120 rpm.

The lignin peroxidase and manganese peroxidase activities are measuredat optimum production in the culture medium, namely at 4.5 days ofculture.

The MnP/LiP ratio is calculated. The results are shown in Table IIIbelow.

                  TABLE III                                                       ______________________________________                                        NAT89                                                                         CONCENTRATION                                                                              MnP ACTIVITY                                                                              LiP ACTIVITY                                                                              MnP/LiP                                  (g/l)                (U/1)                                                                                    (U/1)                                                                                     RATIO                             ______________________________________                                        0.5         6564        1932        3.4                                       0.98                  6780                                                                                     1920                                                                                      3.53                             1.44                  8664                                                                                     1056                                                                                      8.21                             1.89                  6474                                                                                     384                                                                                        16.86                           ______________________________________                                    

III--Production of Lignin Peroxidases and Manganese Peroxidases in aReactor

EXAMPLE 4

Culture in a Reactor Containing Cells Immobilized on a Support

The body of the bioreactor contains the immobilization support, whichconsists in this case of 2 concentric cylinders made of metal gauze(wire 0.15 mm in diameter) having a 0.5 mm mesh, respective diameters of70 mm (outer cylinder) and 30 mm (inner cylinder) for a height of 290mm.

The composition of the basal medium is as follows:

Glycerol 12.5 g/l

Disodium tartrate 2.875 g/l

Diammonium tartrate 2.302 g/l

KH₂ PO₄ 2.5 g/l

CaCl₂.2H₂ O 0.175 g/l

MgSO₄.7H₂ O 0.875 g/l

FeSO₄.7H₂ O 0.0875 g/l

ZnSO₄.7H₂ O 0.0575 g/l

MnSO₄.H₂ O 0.0437 g/l

CUSO₄.5H₂ O 0.0087 g/l

Yeast extract 1.25 g/l

NAT89 0.625 g/l

2.5 liters of this medium are introduced into the bioreactor, and thewhole is sterilized by autoclaving for 30 min at 120° C.

The bioreactor is then thermostated at 37° C. and is aerated withfiltered atmospheric air, introduced at a flow rate of 40 l/h by meansof a circular aeration rod or a sinter forming a steady stream ofbubbles, located at the base of the metal cylinders, providing both foragitation and for a homogeneous aeration needed for the formation of asatisfactory mycelial film.

Inoculation is carried out using mycelial fragments from a preculture.

After 48 hours of culture:

A) The medium is supplemented under sterile conditions with aphospholipid mixture (NAT89) to which veratryl alcohol (VeA) is added.

Composition of the mixture:

VeA 1.25 g

NAT89 0.30 g

B) The stream of air bubbles is replaced by a stream of pure oxygenbubbles at a flow rate of 20 l/h.

The manganese peroxidase and lignin peroxidase activities obtained underthese conditions with the strain MIC 390 are:

maximum MnP activity: 10000 U/l

maximum LiP activity: 2400 U/l.

EXAMPLE 5

Culture of the Microorganism in a Reactor Containing Free Cells, of theBubble Column Type

The composition of the basal medium is as follows:

Glycerol 6.8 g/l

Disodium tartrate 1.565 g/l

Diammonium tartrate 1.252 g/l

KH₂ PO₄ 1.334 g/l

CaCl₂.2H₂ O 0.094 g/l

MgSO₄.7H₂ O 0.467 g/l

FeSO₄.7H₂ O 0.0461 g/l

ZnSO₄.7H₂ O 0.0310 g/l

MnSO₄.H₂ O 0.0232 g/l

CuSO₄.5H₂ O 0.0046 g/l

Yeast extract 0.680 g/l

NAT89 0.5 g/l

2.5 liters of this medium are introduced into the bioreactor, and thewhole is sterilized by autoclaving for 30 min at 120° C.

The bioreactor is then thermostated at 37° C. and aerated with filteredatmospheric air, introduced at a flow rate of 40 l/h by means of acircular aeration rod or a sinter forming a steady stream of bubbles,providing both for agitation and for a homogeneous aeration needed forthe formation of mycelial balls.

Inoculation is carried out using mycelial fragments from a preculture ora solution of spores containing 2×10⁵ spores/ml.

After 24 h, the mycelial balls 0.5 to 1 mm approximately in diameter areformed, and the air flow rate is reduced to 20 1/h.

After 48 h:

A) The medium is supplemented under sterile conditions with aphospholipid mixture comprising veratryl alcohol (VeA).

Composition of the mixture:

VeA 1.05 g

phospholipid source 0.25 g

B) The stream of air bubbles is replaced by a stream of pure oxygenbubbles at a flow rate of 20 l/h.

The results obtained under these conditions with the strain MIC 390 are:

maximum MnP activity: 3600 U/l

maximum LiP activity: 162 U/l.

We claim:
 1. A biologically pure Phanerochaete chrysospoirium strainhaving CNCM number I-1511, I-1512 or I-1513.
 2. A process for producinglignin peroxidase and/or manganese peroxidase from a culture ofPhanerochaete chrysosporium, comprising culturing at least onePhanerochaete chrysosporium strain selected from the group consisting ofCNCM number I-1511 1-1512 and I-1513.
 3. The process according to claim2, wherein the culture medium of Phanerochaete chrysosporium issupplemented during culture by adding veratryl alcohol at aconcentration of between 0.1 and 1 g/l, and/or a phospholipid sourcerich in phosphatidylinositol in the proportion of 0.1 to 3 g/l.
 4. Theprocess according to claim 3, wherein the supplementation withphospholipids and/or with veratryl alcohol is performed afterapproximately 2 days of culture at a temperature of between 28° C. and40° C.
 5. The process according to claim 2, including the preferentialincrease either of the production of manganese peroxidase (MnP) or ofthe production of lignin peroxidase (LiP), as follows:to modify theMnP/LiP ratio in favour of the production of manganese peroxidase, theculturing of Phanerochaete chrysosporium is carried out for a period ofmore than 24 hours and less than 90 hours in the presence of a mixtureof phospholipids at a concentration of between 0.5 and 5 g/l; to modifythe MnP/LiP ratio in favour of the production of lignin peroxidase, theculturing of Phanerochaete chrysosporium is carried out for a period ofmore than 90 hours and less than 350 hours in the presence of a mixtureof phospholipids whose concentration is between 0.5 and 5 g/l.
 6. Theprocess according to claim 2, wherein a culture of cells immobilized ona support is used.
 7. The process according to claim 2, wherein aculture of free cells is used.